March 21, 2008

ABSTRACT While solar light is indispensable for sustenance of life, excessive exposure can cause several skin-related disorders. The UV part of solar radiation, in particular, is linked to disorders ranging from mild inflammatory effects of the skin to as serious as causing several different types of cancers. Changes in lifestyle together with depletion in the atmospheric ozone layer during the last few decades have led to an increase in the incidence of skin cancer. Skin cancers consisting of basal and squamous cell carcinomas are especially linked to the UVB part of solar radiation. Reducing excessive exposure to solar radiation is desirable; however, as this approach is unavoidable, it is suggested that other novel strategies be developed to reduce the effects of solar radiation to skin. One approach to reduce the harmful effects of solar radiation is through the use of phytochemicals, an approach that is popularly known as "Photochemoprotection." In recent years many phytochemicals with potential antioxidant properties have been identified and found to be photoprotective in nature. We describe here some of the most popular phytochemicals being studied that have the potential to reduce the harmful effects associated with solar UV radiation.

INTRODUCTION

During the past few decades, anthropogenic depletion of the stratospheric ozone layer has led to a significant increase in the amount of solar UV radiation reaching the earth's surface. This has led to a significant increase in the amount of UV radiation that people receive. A 1% decrease in the ozone layer is estimated to increase UVB radiation by 2% (1). Compounded by changes in lifestyle related to excessive outdoor recreational activities, UV radiation has consequently led to a surge in the incidence of skin-related disorders including cancer. In the United States alone, more than one million cases of skin cancer are diagnosed each year, accounting for almost 40% of all new cancer cases (2,3). Many countries with a predominantly Caucasian population report similar trends. The most serious form of skin cancer is melanoma which is expected to be diagnosed in about 59 940 patients during the year 2007. Nonmelanoma skin cancers consisting of basal cell carcinomas (BCC) and squamous cell carcinomas (SCC) are, however, the most frequently diagnosed cutaneous malignancies and account for 80% and 16% of all skin cancers, respectively, whereas malignant melanomas account for 4% of all skin cancers. An estimated 8110 deaths are expected to occur as a result of melanomas in the year 2007, while 2740 deaths are expected to occur from nonepithelial skin cancers (4).

Exposure of human or mouse skin to UVB radiation results in excessive generation of reactive oxygen species (ROS) that overwhelms the antioxidant defense system resulting in oxidative stress (2,5,6). Several skin-related disorders such as photoaging and photocarcinogenesis are believed to be mediated by the generation of ROS (7,8). The acute responses of UV radiation are DNA damage, lipid peroxidation and protein crosslinking that lead to erythema, sunburn and immunosuppression. Chronic UV responses result in dysregulation of apoptosis leading to abnormal proliferation of keratinocytes containing DNA damage, acquisition of p53 mutations and alterations in signal transduction pathways, all of which contribute to the onset of skin cancer (9). UV irradiation results in the induction of matrix metalloproteinases (MMPs), which degrade the collagen and connective tissue components of the skin. Recent studies suggest that UV radiation blocks transforming growth factor- beta Type II receptor/Smad signaling (10), activates activator protein (AP)-1 that in turn is activated by a series of mitogen- activated protein kinases (MAPKs). In addition, nuclear factor kappa B (NF-kB), a transcription factor, is also activated by UV irradiation. It is suggested that both AP-1 and NF-kappaB that are activated by ROS may provide the complex driving force that results in a series of complex biologic interactions leading to skin- related diseases, most notably skin cancer (2,3,11,12).

As trends related to exposure of humans to solar UV radiation are expected to continue in the near future and pose a substantial human health risk, adverse effects associated with UVB exposure need to be appropriately addressed. While avoiding excessive exposure to sun, wearing protective clothing and using sunscreen lotions are popular recommendations, there is a need for the development of effective phytochemicals that are capable of ameliorating the adverse effects of UVB. The discovery that various plant-derived phytochemicals possess photoprotective properties is hopeful. Some of these agents- popularly known as "photochemopreventive agents"-are also present in the human diet and many such agents have also found a place in various skin care products. Understanding how such phytochemicals exert their effects is not only important but essential to the development of better and more effective photochemopreventive products for general human use.

Green tea polyphenols were for the first time identified to afford protection against UVB radiation-induced carcinogenesis in an elegant study emanating from the laboratory of Prof. Hasan Mukhtar (13). A series of subsequent studies led to the identification of other phytochemicals and the mechanisms associated with the inhibition of photocarcinogenesis (2,8,14-17). Modulations in NF- kappaB and MAPKs pathways have been identified as a molecular basis for the photochemopreventive effects of green tea constituent (-)- epigallocatechin-3-gallate (EGCG) (18,19). The purpose of this review was to summarize work on the use of phytochemicals for prevention of damages associated with exposure to solar UV radiation. Discussion of the adverse effects of UVB radiation is followed by the photoprotective and photochemopreventive potentials of some of the most popular phytochemicals present in diet and beverages.

ULTRAVIOLET RADIATION-INDUCED SKIN CANCER OR PHOTOCARCINOGENESIS

Solar UV radiation-induced skin cancer or photocarcinogenesis is a complex multistage phenomenon involving three distinct stages exemplified by initiation, promotion and progression (2,3). Each of these stages is mediated via alterations at various cellular, biochemical and molecular levels. Initiation, the first step in the carcinogenesis process, is an irreversible step in which genetic alterations occur in genes that ultimately leads to DNA mutation in normal cells. Tumor promotion is the vital process in cancer development involving clonal expansion of initiated cells giving rise to premalignant and then to malignant lesions, essentially by alterations in signal transduction pathways. Tumor progression involves the conversion of premalignant and malignant lesions into an invasive and potentially metastatic malignant tumor (2,3). To name a few, the process of skin cancer development involves stimulation of DNA synthesis, DNA damage, cell proliferation, inflammation, epidermal hyperplasia, immunosuppression, cell cycle dysregulation, depletion of antioxidant enzymes, impairment of signal transduction pathways, induction of ornithine decarboxylase (ODC) and cyclooxygenase-2 (COX-2) (2,20,21).

ULTRAVIOLET RADIATION AND DNA DAMAGE

DNA contains purine and pyrimidine bases and their absorption maxima lie between 230 and 300 nm. Therefore, DNA is a major UVB- absorbing cellular chromophore in the skin. The most common and frequent photoproducts that are formed in the skin after UVB exposure are cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6- 4) pyrimidone photodimers. UVB irradiation to the skin also results in DNA strand breaks, DNA crosslinks and DNA-protein crosslinks (2,9). Formation of CPDs, if not repaired through nucleotide excision repair, leads to signature mutations. These mutations are in the form of C to T and CC to TT transition and have been observed in tumor suppressor gene p53 from specimens of human SCCs, BCCs and actinic keratoses. Studies suggest that most of the UVB-induced CPDs are found in the epidermis but a significant amount is also found in the dermis. Pharmacokinetic studies reveal that UVB-induced DNA damage in the form of CPDs in human skin declines with time because the damage is either repaired or the damaged cells undergo apoptosis (22). CPDs are primarily produced in keratinocytes and Langerhans cells following UVB exposure, and also in dendritic cells of lymph nodes draining the irradiated sites. CPDs have been implicated in UVB-induced immunosuppression and initiation of photocarcinogenesis (2,23). The p53 tumor suppressor gene plays a decisive role in protecting cells from DNA damage as a consequence of UVB exposure (2,9,24). It has also been suggested that p53 can play direct and indirect roles in UVB-induced, transcription-coupled DNA repair (25). The increased level of p53 protein after DNA damage is also associated with enhanced apoptosis, presumably in those cells that are too damaged for adequate DNA repair (9,26). High doses of UVB have been shown to be associated with the formation of sunburn cells, initiated via a p53-dependent pathway, which causes the removal of damaged cells, thus minimizing the risk of skin cancer (27). Lower doses of UVB allow cell survival and repair of genetic damage, but the cell survival response is a state of local and systemic immunosuppression, which in itself may be deleterious and contribute to the process of formation of skin cancers (28). ULTRAVIOLET RADIATION AND IMMUNOSUPPRESSION

UVB exposure initiates a cascade of events that modify gene expression profiles and alters the immune system of the skin (21). There is ample experimental and clinical evidence to suggest that immune factors contribute to the pathogenesis of solar UV-induced skin cancer (20,21). Chronically immunosuppressed patients living in regions of intense sun exposure experience an exceptionally high rate of skin cancer. It is generally documented that UVB initiates immunosuppression through three possible photoreceptors: DNA damage, the isomerization of trans-urocanic acid (UCA) to cis-UCA, or lipid membrane peroxidation. Exposure to UVB radiation results in inhibition of contact hypersensitivity (CHS) induced by contact allergens that is a prototypic T-cell-mediated immune response (29,30). Interleukin-12 (IL-12) can be produced locally in the skin either by keratinocytes or by macrophages (31,32). UVB-induced infiltration of CD11b^sup +^ cells (cell surface markers of monocytes/macrophages and neutrophils) upregulates IL-10 and downregulates IL-12 in the skin and/or draining lymph nodes (33). IL- 10 possesses immunosuppressive activity (34), whereas IL-12 is an immunoregulatory cytokine (35). In UVB-exposed skin, IL-10 is primarily secreted by activated macrophage and inhibits antigen presentation, thereby downregulating CHS responses (36). IL-12 by stimulating the production of interferon-gamma regulates the growth of T cells and enhances T helper cell Type-1 function (35,37). IL- 12 suppresses UV-induced apoptosis by inducing DNA repair both in vitro and in vivo (38).

ULTRAVIOLET RADIATION-INDUCED AGING OR PHOTOAGING

Skin aging is a dynamic, multifactorial process consisting of two distinct components: (1) intrinsic or natural aging, genetically determined degenerative aging processes, is unavoidable and results in cutaneous alterations, and (2) extrinsic aging that also manifests in cutaneous changes, originates from exogenous sources and is avoidable. The process of aging, both intrinsic and extrinsic, is influenced by the formation of ROS. Extrinsic aging results from various factors, but exposure to solar UV radiation is the primary cause. Depending on the amount and form of the UVB radiation, as well as on the skin type of the individual exposed, UVB irradiation causes premature skin aging, referred to as photoaging (39). Solar UV radiation has a profound effect on exposed skin, producing accelerated age-related changes consisting of fine and coarse wrinkling, rough skin texture, dryness, telangiectasia, and dyspigmentation abnormalities including lentigines as well as guttate hypermelanosis and hypomelanosis (40). Studies have suggested that there is an increased generation of ROS in skin upon UV exposure. Increased ROS generation can overwhelm antioxidant defense mechanisms, resulting in oxidative stress and oxidative photodamage of proteins and other macromolecules in the skin. DNA photodamage and UV-mediated generation of ROS are the initial molecular events that lead to most of the typical histologic and clinical manifestations of chronic photodamage of the skin. These events are believed to be critical mediators of the photoaging process (8,41). ROS can modify proteins in tissue to form carbonyl derivatives, which accumulate in the papillary dermis of photodamaged skin (42). Mast cells and macrophages are found in greater numbers in photodamaged skin and are believed to be involved in photoaging (43). Exposure of human or mouse skin to UV irradiation induces MMPs, which have been implicated in photoaging (44). UV radiation activates AP-1 that stimulates transcription of MMP genes encoding MMP-1 (collagenase), MMP-9 (gelantinase) and MMP- 3 (stromelysin-1) in skin cells. These changes apparently occur through induction of AP-1 that is activated by a series of MAPKs. Together, these MMPs are capable of degrading the collagen framework and other components of skin connective tissue (44). Studies have suggested that solar UV reduces collagen in photoaged human skin by blocking transforming growth factor-beta Type II receptor/Smad signaling (10). In addition, NF-kappaB, a transcription factor, is activated by UV irradiation, which stimulates neutrophil attraction bringing neutrophil collagenase (MMP-8) into the irradiation site to further aggravate matrix degradation. Both AP-1 and NF-kappaB are activated by ROS that may provide the common denominator for driving these complex biologic interactions (11,12). Oxidative stress increases elastin mRNA levels in dermal fibroblasts resulting in elastotic changes found in photoaged dermis (12).

PHYTOCHEMICALS FOR PHOTOCHEMOPREVENTION

In recent years, phytochemicals present in the common diet consumed by the human population have gained considerable attention as photoprotective and photochemopreventive agents against skin cancers (2,7). Studies have shown that phytochemicals present in the human diet and in beverages afford protection against the development of cutaneous malignancies and other skin disorders (2,7). Epidemiologic and experimental studies have revealed that a wide variety of phytochemicals can alter or correct undesired cellular functions, leading to photoprotective effects. These phytochemicals belong to several classes that include polyphenols, flavonoids, isoflavonoids, phytoalexins, phenols, anthocyanidins and carotenoids. Phytochemicals may work in different ways: by stimulating the immune response, by inducing gene suppression, by blocking oxidative damage to DNA, by detoxifying carcinogens and by initiating selected signaling pathways or by other mechanisms. It is becoming clear that many of these phytochemicals play multiple roles in ameliorating the process of photocarcinogenesis. Thus, the photochemopreventive approach appears to have practical implications in reducing skin cancer risk as individuals can modify their dietary habits and lifestyle in combination with careful use of skin care products. Table 1 summarizes the biologic effects and postulated mechanism(s) of individual phytochemicals in relation to their photochemopreventive potential.

EGCG

EGCG, the major and the most active polyphenols constituent found in green tea (dried fresh leaves of the plant Camellia sinensis L. [Theaceae]), is one of the most extensively investigated phytochemicals for photoprotection. EGCG acts as a potent antioxidant and can scavenge ROS, such as lipid free radicals, superoxide radicals, hydroxyl radicals, hydrogen peroxide and singlet oxygen. We found that treatment of normal human epidermal keratinocytes (NHEK) with EGCG resulted in inhibition of UVB- mediated phosphorylation and degradation of IkappaBalpha, activation of IKKalpha and NF-kappaB, and phosphorylation of MAPK in a dose- and time-dependent manner (18,19). Our data suggest that EGCG protects against the adverse effects of UV radiation via modulations in NF-kappaB and MAPK pathways. Kim et al. (45) have shown that EGCG inhibits UVB-induced NF-kappaB binding activity both under in vivo and in vitro situations. Treatment of HaCaT cells with EGCG inhibited UVB induced c-fos gene expression and AP-1 activation (46). Further, using the human keratinocyte cell line HaCaT, it was shown that EGCG is effective in inhibiting AP-1 activity when these cells were treated before, after or both before and after UVB irradiation (47). Pretreatment of mouse epidermal JB6 C141 cells with EGCG inhibited UVB-induced phosphatidylinositol-3 kinase activation (48).

Topical application of EGCG to C3H/HeN mice before a single dose of UVB exposure decreased H^sub 2^O^sub 2^-producing cells, inducible nitric oxide synthase-expressing cells and also decreased the production of H^sub 2^O^sub 2^ and NO in both epidermis and dermis at the UVB-irradiated site (49). EGCG protects against UV- induced oxidative stress in humans as well. When it was applied to the skin of volunteers just before exposure to a 4 x minimal erythema dose (MED) of UVB radiation, it significantly decreased the production of hydrogen peroxide and nitric oxide production as well as lipid peroxidation in the dermis and epidermis (50). EGCG pretreatment also inhibits UV-induced infiltration of inflammatory leukocytes, particularly CD11b^sup +^ cells (a surface marker of monocytes/ macrophages and neutrophils), into the skin and these cells are considered to be the major producers of ROS. A single UV exposure of 4 x MED to human skin was found to increase catalase activity and decrease glutathione peroxidase (GPx) activity and total glutathione (GSH) level at different time points studied. Pretreatment with EGCG was found to restore the UVB-induced decrease in GSH level and afforded protection to the antioxidant enzyme GPx (50).

Table 1. Phytochemicals for protection against UVB-mediated damages.

Table 1. Phytochemicals for protection against UVB-mediated damages.

Conney et al. (51) reported that oral administration of EGCG inhibits the growth of well-established skin tumors and, in some cases, tumor regression was also observed. In mice, systemic and topical administration of EGCG was found to protect against the UV- induced sunburn response (33). In contrast to its effect on normal keratinocytes, EGCG stimulates apoptosis in UV-induced premalignant papillomas and invasive SCC in mice (52). Administration of EGCG in drinking water significantly decreased tumor number and total tumor burden compared with untreated ODC/Ras mice without decreasing the elevated polyamine levels present in the ODC/Ras mice. This ODC/Ras double transgenic mouse model develops spontaneous skin tumor because of overexpression of ODC and a v-Ha-ras transgene. Many epithelial tumors exhibit aberrant Ras signaling either due to ras mutations or mutations in other genes that can also lead to chronic upregulation of the Ras pathway. EGCG selectively decreased both proliferation and survival of primary cultures of ODC over- expressing transgenic keratinocytes but not keratinocytes from normal littermates or ras-infected keratinocytes (53). Topical treatment of SKH-1 hairless mice with EGCG in a hydrophilic ointment- based formulation resulted in exceptionally strong inhibition in tumor incidence (60%), tumor multiplicity (86%) and tumor growth, in terms of total tumor volume (95%) per group (54). These results indicate that the use of EGCG in topical formulation might increase penetration or absorption capacity inside the skin layers. Topical application of EGCG to the mouse skin prior to UV irradiation was shown to result in inhibition of CHS response to contact sensitizer, reduction in the number of infiltrating macrophages (CD11b^sup +^ cells) and neutrophils, downregulation in UV-induced production of IL-10 and increased production of IL-12 in the skin and draining lymph nodes (33). EGCG was also found to balance the alterations in the IL-10/IL-12 cytokines. This effect may be mediated by the antigen-presenting cells in the skin and draining lymph nodes or by blocking the infiltration of IL-10-secreting CD11b^sup +^ macrophages into the irradiated site (33). Recently, Meeran et al. (55) have shown that topical application of EGCG prevented UV- induced suppression of CHS in wild-type (WT) mice, as shown by significant enhancement of CHS response (ear swelling). In contrast, UV-exposed IL-12 KO mice remained unresponsive to 2,4- dinitroflurobenzene despite the application of EGCG on mouse skin, indicating that the immunopreventive effect of EGCG on UV-induced suppression of CHS requires IL-12 or is mediated through IL-12. Further, in EGCG-treated mice, i.p. injection of anti-IL-12 antibody significantly reversed or blocked the preventive effect of EGCG on the UV-induced suppression of CHS. These studies suggest that the prevention of UV-induced suppression of CHS by EGCG is mediated, at least in part, through IL-12 (55). Topical application of EGCG significantly inhibited tumor incidence, tumor multiplicity and tumor growth or tumor size in WT (C3H/HeN) mice, but not in IL-12 KO mice, indicating that the prevention of UVB-induced skin cancer by EGCG requires IL-12 (56).

Treatment of cultured cells with EGCG directly inhibits the expression of MMPs such as MMP-2, MMP-9 and neutrophil elastase even in the absence of UV exposure (57). Chronic exposure of mouse skin to UVB has been shown to induce the expression of MMP-2, -3, -7 and - 9, which are involved in the degradation of Types-I and -III collagen fragments generated by collagenases (58), and Type IV collagen of the basement membrane (59). Treatment of EGCG diminishes UVA-induced skin damage (roughness and sagginess) and protects from the loss of dermal collagen in hairless mouse skin, and also blocks the UV-induced increase in collagen secretion and collagenase mRNA level in cultured human epidermal fibroblasts and the promoter- binding activities of AP-1 and NF-kappaB (45).

Genistein

Genistein (5,7,4'-trihydroxyisoflavone) is an isoflavone that was first isolated from soybean (60). It displays a very low level of toxicity in most animal species. Although soybean contains a number of ingredients with demonstrated anticancer activities, genistein is the most important agent that has been extensively investigated for its chemopreventive and anticancer activity (61). Genistein has been shown to potently inhibit the activities of tyrosine protein kinase (TPK), topoisomerase II and ribosomal S6 kinase in the cell culture (62). Genistein specifically inhibits the growth of ras oncogene- transfected NIH 3T3 cells without affecting the growth of normal cells and diminishes the c-fos and c-jun expression in CH310T1/2 fibroblasts induced by platelet-derived growth factor (63).

Resveratrol (trans-3,4',5-trihydroxystilbene) is a polyphenols phytoalexin found largely in the skin and seeds of grapes, nuts, fruits and red wine. Resveratrol is a potent antioxidant with anti- inflammatory and antiproliferative properties (68,69). Single topical application of resveratrol to SKH-1 hairless mice prior to UVB irradiation resulted in significant inhibition of UVB-induced skin edema and caused a significant decrease in UVB-mediated generation of H^sub 2^O^sub 2^ and infiltration of leukocytes (68). Resveratrol treatment to mouse skin was also found to result in significant inhibition of UVB-mediated induction of COX and ODC enzyme activities and protein expression, which are well- established markers for tumor promotion. It was also observed that resveratrol inhibited UVB-mediated increased level of lipid peroxidation, a marker of oxidative stress (68). In another study, pretreatment of NHEK with resveratrol inhibited UVB-mediated activation of the NF-kappaB pathway (69). Resveratrol blocked UVB- mediated activation of NF-kappaB in NHEK in a dose- and time- dependent fashion. Resveratrol treatment of NHEK also inhibited UVBmediated phosphorylation and degradation of IkappaBalpha, and activation of IKKalpha (69). Studies have demonstrated that resveratrol imparts its protective effect against multiple UVB exposure via modulations in the cki-cyclin-cdk network and the MAPK pathway (70). Further, in short-term experiments, topical application of resveratrol to SKH-1 hairless mouse skin prior to UVB irradiation resulted in significant inhibition of UVB-induced cellular proliferation, protein and mRNA levels of survivin, phosphorylation of survivin and upregulation of proapoptotic Smac/ DIABLO protein (71). In long-term studies, topical application of skin with resveratrol (both pre- and posttreatment) has been shown to result in significant inhibition in tumor incidence and delay in the onset of tumorigenesis (72). Both post- and pretreatments were equally effective, suggesting that resveratrol-mediated responses may not be sunscreen effects.

Silymarin

Silymarin, a polyphenols flavonoid isolated from milk thistle plant (Silybum marianum L. Gaertn), is a mixture of several flavonolignans, which includes silybin, silibinin, silidianin, silychristin and isosylibin (73). The antioxidant and anticarcinogenic effects of silymarin in mouse models of chemical and photocarcinogenesis have been established, and silybin has been shown to be the main constituent responsible for these effects (74,75). It has been shown recently that treatment of irradiated HaCaT cells with Silymarin resulted in concentration-dependent diminution of UVA-caused oxidative stress. Silymarin application extensively reduced GSH depletion and ROS production as well as lipid peroxidation in irradiated cells. Formation of UVA-induced DNA single strand breaks and caspase-3 activity was also significantly decreased by Silymarin (76). Silibinin restored UVB-caused depletion of survivin, concomitant with upregulation of NF-kappaB DNA binding activity. Further, silibinin treatment upregulated UVB-induced extracellular signal-related kinase 1/2 (ERK 1/2) phosphorylation and increased duration of the S phase, possibly providing a prolonged time for efficient DNA repair (77). Treatment of C3H/HeN mice with topically applied Silymarin or silibinin, a major component of Silymarin, markedly inhibited UVB-induced suppression of CHS response in a local model of immunosuppression and had a moderate inhibitory effect in a systemic model of CHS. Silymarin reduced the UVB-induced enhancement of the levels of the immunosuppressive cytokine, IL-10, in the skin and draining lymph nodes and enhanced the levels of the immunostimulatory cytokine, IL- 12 (78). Dietary feeding of silibinin to SKH-1 hairless mice for 2 weeks before a single UVB irradiation resulted in a strong and significant decrease in UVB-induced CPD^sup +^ cells and PCNA, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and apoptotic sunburn cells together with an increase in p53 and p21/cip1-positive cell population in the epidermis (79). Silibinin strongly prevented UVB-induced apoptosis, as observed by a reversal in UVB-caused PARP cleavage, caspase-9 activation and an increase in apoptotic cells. Topical treatment of silymarin to C3H/HeN mice inhibits UVBinduced suppression of CHS response to the contact sensitizer dinitrofluorobenzene. Prevention of UVB-induced suppression of CHS by silymarin was found to be associated with the inhibition of infiltrating leukocytes, particularly the CD1b^sup +^ cell type, and myeloperoxidase activity. Silymarin treatment also resulted in a significant reduction in UVB-induced immunosuppressive cytokine IL-10 producing cells and its production. Topical treatment of silymarin also resulted in a significant reduction in the number of UVB-induced H^sub 2^O^sub 2^producing cells and inducible nitric oxide synthase- expressing cells concomitant with a decrease in H^sub 2^O^sub 2^ and nitric oxide production (80). Topical application of silymarin protects against photocarcinogenesis in mice. SKH-1 hairless mice were subjected to (1) tumor initiation with UVB, followed by tumor promotion with tetradecanoylphorbol-13-acetate, (2) tumor initiation with 7,12-dimethylbenz[a]anthracene (DMBA), followed by tumor promotion with UVB and (3) tumor initiation and promotion with UVB. In all three groups, topical application of silymarin prior to exposure to UVB or DMBA significantly reduced tumor incidence, tumor multiplicity and average tumor volume per mouse. Furthermore, in short-term experiments, the topical application of silymarin was found to result in significant inhibition against UVB-induced (1) skin edema, (2) skin sunburn and cell apoptosis, (3) depletion of catalase activity and (4) induction of COX-2 and ODC activities and mRNA expression. These studies suggest that topical application of silymarin provides substantial protection against UVB-mediated damage in mouse skin, possibly via its strong antioxidant properties at different stages of UVB-induced carcinogenesis (81).

Apigenin

Apigenin (5,7,4'-trihydroxyflavone) is a natural flavonoid present in the leaves, stems, fruits and vegetables of vascular plants. A beneficial role of flavonoids in the treatment and prevention of skin disorders has been suggested. Initial studies showed that apigenin treatment to murine skin resulted in inhibition of UV-mediated induction of ODC activity as well as reduction in cancer incidence with an increase in tumor-free survival (82). In addition, delivering apigenin into viable epidermis seemed to be essential for an apigenin formulation to be effective in skin cancer prevention (83) as apigenin strongly absorbs UV light, with three maximum absorption wavelengths at 212, 269 and 337 nm (84).

Lepley et al. (85) examined the effects of apigenin on the cell cycle and indicated that apigenin induced a reversible G2/M arrest in cultured keratinocytes, partly through inhibition of the mitotic kinase activity of p34cdc2, and perturbation of cyclin B1 levels. Subsequent studies in human diploid fibroblasts provided evidence that apigenin can induce G1 arrest in addition to arresting cells at G2/M phase of the cell cycle. This was accompanied by inhibition of cdk2 kinase activity, phosphorylation of the retinoblastoma protein and induction of the (cyclin-dependent kinase) cdk inhibitor p21/ waf1, all of which may mediate its chemopreventive activities in vivo (86). Additional data suggested that apigenin may exert antitumorigenic activity by stimulating the p53-p21/waf1 response pathway. These findings were reinforced by the observation that apigenin-induced G2/M arrest of cultured mouse keratinocytes was accompanied by an increase in the p53 protein stability in addition to p21/waf1 expression (87). In addition, apigenin had little effect on the accumulation of cyclin B1 protein (88). Recently it was shown that apigenin can prevent photocarcinogenesis by inhibiting the induction of COX-2 protein expression in mouse keratinocytes. Apigenin is thought to inhibit UV-induced COX-2 expression through modulation of upstream stimulatory factor (USF) transcriptional activity in the 5' upstream region of the COX-2 gene. Additionally, it suppressed the translation of the RNA-binding protein, T-cell- restricted intracellular antigen 1-related protein (TIAR) and augmented the localization of TIAR and HuR in the cytoplasm. More importantly, reduction in HuR levels by small interfering RNA was shown to inhibit apigenin-mediated stabilization of COX-2 mRNA (89).

Naturally occurring flavonoids including apigenin have been shown to inhibit MMP-1 activity and downregulate MMP-1 expression via an inhibition of AP-1 activation, although the cellular inhibitory mechanisms differ depending on their chemical structures (90). The structure-activity relationship of the antioxidative property of flavonoids was studied by Sim et al. (91) and it was found that the inhibitory effects of flavonoids on collagenase in human dermal fibroblasts depends on the number of OH group in the flavonoid structure, and those with a higher number of OH group may be more useful in protecting the skin from photoaging.

Curcumin

Curcumin, one of the most studied chemopreventive agents, is a natural compound extracted from Curcuma longa L. that allows suppression, retardation or inversion of carcinogenesis. Curcumin is also described as an antitumoral, antioxidant and anti-inflammatory agent capable of inducing apoptosis in numerous cellular systems. The varied biologic properties of curcumin and lack of toxicity even when administered at higher doses makes it attractive to explore its use in various disorders such as tumors of the skin, colon, duodenum, pancreas, breast and other skin diseases. The molecular basis of anticarcinogenic and chemopreventive effects of curcumin is attributed to its effect on several targets, including transcription factors, growth regulators, adhesion molecules, apoptotic genes, angiogenesis regulators and cellular signaling molecules (92).

It has been shown recently that on treatment of HaCaT cells with UVB and curcumin, there was induction of apoptosis as demonstrated by DNA fragmentation. Combination of UVB irradiation with curcumin also synergistically induced apoptotic cell death in HaCaT cells through activation of caspase-8, -3 and -9 followed by release of cytochrome c (93). Treatment with curcumin strongly inhibited COX-2 mRNA and protein expressions in UVB-irradiated HaCaT cells. Notably, there was effective inhibition by curcumin on UVB-induced activations of p38 and JNK in HaCaT cells. The DNA-binding activity of AP-1 transcription factor was also markedly decreased with curcumin treatment in UVB-irradiated HaCaT cells. These results collectively suggest that curcumin may inhibit COX-2 expression by suppressing p38 and JNK activities in UVB-irradiated HaCaT cells (94). It has been found that topical application of curcumin in TPA- pretreated epidermis of CD-1 mice significantly inhibited UVA- induced ODC activity and reduced TPA-induced dermatitis (95). Topical application of curcumin significantly inhibited both TPA and UVA-induced ODC and metalloprotein gene expression. The inhibitory effects of curcumin were attributed to its ability to scavenge ROS by interrupting the activation of protein kinase C (96).

[6]-Gingerol

[6]-Gingerol, a pungent ingredient of ginger (Zingiber officinale Roscoe, Zingiberaceae), has antibacterial, anti-inflammatory and antitumor-promoting activities. It has been found to possess substantial antioxidative activity as determined by inhibition of phospholipid peroxidation induced by the FeCl^sub 3^ascorbate system (97). [6]-Gingerol also exerts an inhibitory effect on xanthine oxidase (98) which is responsible for the generation of ROS, such as superoxide anion. More recently, Guh et al. (99) reported concentration-dependent inhibition by [6]-gingerol of arachidonic acid-induced platelet aggregation and formation of thromboxane B^sub 2^ and prostaglandin D^sub 2^. The ethanol extract of ginger reduced carrageenan-induced paw edema (100).

It has been reported recently that pretreatment with [6]gingerol reduced UVB-induced intracellular ROS levels, activation of caspase- 3, -8, -9 and Fas expression. It also reduced UVB-induced expression and transactivation of COX-2. Translocation of NF-kappaB from the cytosol to the nucleus in HaCaT cells was inhibited by [6]-gingerol via suppression of IkappaBalpha phosphorylation. It was also shown that topical application of [6]-gingerol prior to UVB irradiation of hairless mice also inhibited the induction of COX-2 mRNA and protein, and NF-kappaB translocation (101).

Delphinidin

Delphinidin, a dietary anthocyanidin present in many pigmented fruit and vegetables such as pomegranates, berries, dark grapes, egg plants, tomatoes, carrots, purple sweet potatoes and red onions, possesses strong antioxidant, anti-inflammatory and antiangiogenic properties (102,103). Anthocyanidins, aglycons of anthocyanins, make a major contribution to the total polyphenol dietary intake (103). Pretreatment of HaCaT cells with delphinidin protected against UVB- mediated (1) decrease in cell viability and (2) induction of apoptosis. Furthermore, pretreatment of HaCaT cells with delphinidin inhibited UVB-mediated (1) increase in lipid peroxidation; (2) formation of 8-OHdG; (3) decrease in PCNA; (4) increase in PARP cleavage; (5) activation of caspases; (6) increase in Bax; (7) decrease in Bcl-2; (8) upregulation of Bid and Bak; and (9) downregulation of BclxL. Topical application of delphinidin to SKH- 1 hairless mouse skin inhibited UVB-mediated apoptosis and markers of DNA damage such as CPD and 8-OHdG. These results suggest that treatment of HaCaT cells and mouse skin with delphinidin inhibited UVB-mediated oxidative stress and reduced DNA damage, thereby protecting the cells from UVB-induced apoptosis (102). The relevance of these findings in cell culture system to photocarcinogenesis and photoaging is currently being investigated in our laboratory. Lycopene

Carotenoids are a group of at least 600 compounds produced by plants, and they account for many of the bright colors in the plant kingdom. The most common carotenoids in the human diet and plasma are beta-carotene, alpha-carotene, lycopene, lutein, and beta- cryptoxanthin. Of the 14 carotenoids found in human serum, tomato and tomato products contribute to nine and are the predominant source of about one-half, including lycopene. In fact, in most populations, particularly in the West, dietary lycopene is supplied largely by tomatoes and tomato-based products (104).

Application of lycopene dose-dependently inhibited UVB-induced ODC and myeloperoxidase activities and significantly reduced bifold skin thickness. Application of topical lycopene prevented the cleavage of caspase-3 and significantly reversed UVB-induced PCNA inhibition and normal PCNA staining was restored in the lycopene- treated skin (105). Following ingestion of lycopene or tomato- derived products rich in lycopene, photoprotective effects have been demonstrated. After 10-12 weeks of intervention, a decrease in the sensitivity toward UV-induced erythema was observed in volunteers (106). In a study involving 25 healthy individuals, the capacity of an antioxidant complex (AOC) vitamins (lycopene, beta-carotene, alpha-tocopherol), selenium was observed to reduce UV-induced damages. After the oral intake of AOC, there was an elevation of the actinic erythema threshold and a general reduction in UV-induced erythemas, a reduction in UV-induced p53 expression and sunburn cells, and a parallel reduction in lipoperoxide levels. Pigmentation was also increased (107).

CONCLUSION

The incidence of skin cancer has been rising in recent years with significant effects on public health. Cutaneous damage, premature aging of the skin, and skin cancer ensue when UV exposure exceeds the protective capacity of the antioxidant system. To combat these adverse effects, there is a need to educate people regarding the harmful effects of UV exposure, especially of sunbathing. Primary prevention such as the use of photoprotective clothing and application of sunscreen have proven inadequate in impacting the incidence of skin cancer, thus emphasizing the need for development of photochemo-preventive strategies. The use of skin care products supplemented with a combination of different phytochemicals, working through different mechanisms in conjunction with the use of sunscreens and educational efforts, may be an effective approach for reducing UV-mediated damages.

Acknowledgements-This work was supported by a grant from USPHS Grant R21 AT002429-01. Studies related to the beneficial effects of phytochemicals emanating through the work of the authors have been possible because of their association with Prof. Hasan Mukhtar whose insight into the subject has been a guiding force in the development of many phytochemicals and identifying their potential photochemopreventive properties. As a scientific supervisor and mentor par excellence, Prof. Mukhtar has inculcated in a generation of young investigators the ideals of scientific judgment, survival skills, perseveration and persistence. He continues to impart and pass on his knowledge through challenging discussions both at the bench and during laboratory meetings. As we celebrate his 60th birthday, we would like to express our sincere gratitude to Prof. Mukhtar, and wish many young investigators the good fortune of being in his company.

[dagger] This paper is part of a special issue dedicated to Professor Hasan Mukhtar on the occasion of his 60th birthday.